Octalactone-containing composition, fermentation process for producing same and organoleptic uses thereof

ABSTRACT

Described is a process for the preparation of compositions of matter containing octalactones defined according to the structure: ##STR1## which includes the stereoisomers having the structures: ##STR2## by means of the sequential steps of (i) fermentation of caprylic acid or the ethyl ester of caprylic acid defined according to the generic structure: ##STR3## wherein R is hydrogen or ethyl using a microorganism selected from the group consisting of: 
     Mortierella ramanniana var. ramanniana--CBS 112.08 
     Mortierella ramanniana var. ramanniana--CBS 478.63 
     Choanephora cucurbitarum--NRRL 2744 
     Mortierella isabellina--IFO 8183 
     Mortierella isabellina--IFO 8309 
     Mortierella nana--IFO 8794 
     Mortierella isabellina--IFO 7824 
     Mortierella vinacea--IFO 6738 
     whereby a gamma hydroxy octanoic acid defined according to the structure: ##STR4## is formed; and (ii) lactonization of the resulting gamma hydroxy octanoic acid by means of simultaneous acidification and heating according to the reaction sequence: ##STR5##

RELATED APPLICATIONS

This application is a continuation-in-part of application for U.S. Pat.patent Ser. No. 797,055 filed on Nov. 25, 1991 now U.S. Pat. No.5,166,366 which is a divisional of application for U.S. patent Ser. No.719,154 filed on Jun. 21, 1991 (now U.S. Pat. No. 5,112,803 issued onMay 12, 1992).

BACKGROUND OF THE INVENTION

This invention is concerned with a microbial process for the productionof compositions of matter containing octalactone having the genericstructure: ##STR6##

Considerable time and effort have been expended by microbiologists inthe search for better processes for the production of saturatedlactones; and more generally, lactones per se. U.S. Pat. No. 3,076,750discloses a method for preparing certain optically active lactones andthe corresponding hydroxycarboxylic acids by microbial reduction ofketocarboxylic acids. The metabolism of ricinoleic acid by some Candidastrains was investigated by Okui, et al (J. Biochemistry, 54,536-540,1963) who showed that gamma hydroxy decanoic acid was an intermediate inthe oxidative degradation of ricinoleic acid. However, only traceamounts of gamma hydroxydecanoic acid were recovered from thefermentation medium due to the metabolism of gamma hydroxydecanoic acidupon completion of the fermentation, and the toxicity of ricinoleic acidto the microorganism, which limits the amount of substrate that can beused.

U.S. Pat. No. 4,560,656 provided a method of producing optically activegamma hydroxydecanoic acid comprising culturing or incubating amicroorganism capable of hydrolyzing castor oil, and effectingbeta-oxidation of the resulting hydrolysate in the presence of castoroil, to produce gamma hydroxydecanoic acid.

U.S. Pat. No. 4,560,656 also provided a method of producing opticallyactive gamma hydroxydecanoic acid comprising enzymatically hydrolyzingcastor oil using lipase to form an enzymatic hydrolysate and culturingor incubating a microorganism capable of effecting beta-oxidation of theenzymatic hydrolysate in the presence of said hydrolysate to producegamma hydroxydecanoic acid.

U.S. Pat. No. 4,560,656 also provided a method of producing opticallyactive gamma hydroxydecanoic acid comprising culturing or incubating amicroorganism capable of hydrolyzing castor oil and a microorganismcapable of effecting beta-oxidation of castor oil hydrolysate in thepresence of castor oil to produce gamma hydroxydecanoic acid.

European Published Patent Application 258993 published on Apr. 9, 1988discloses a process for the production of optically active gammahydroxydecanoic acid suitable for conversion to optically active gammadecalactone. The process covers steps of:

(a) culturing Sporobolomyces odorous; and/or Rhodotorula glutinis on amedium containing a ricinoleic acid source at 15°-35° C. at a pH of 3-9and, optionally;

(b) lactonizing the resulting gamma hydroxydecanoic acid to gammadecalactone.

Lion Corporation, European Published Patent Application No. 269,351filed on Nov. 17, 1987 discloses a method for producing a fat containinggamma linolenic acid comprising the steps of culturing a microorganismbelonging to the genus Absidia, the genus Mortierella, the genus Mucor,the genus Rhizopus or the genus Syncephalastrum with a fatty acid or anester thereof as the carbon source, and converting the fatty acid or theester thereof to gamma linolenic acid. The microorganisms exemplifiedare Absidia corymbifera, IFO 4010, Mortierella isabellina, IFO 7873,Rhizopus oryzae, IFO 5418 and Syncephalastrum racemosum, IFO 4816. Fattyacids or esters exemplified are set forth in paragraph 5, on page 3 ofEuropean Application 269,351, to wit, fatty acids having 8 to 22 carbonatoms, particularly 8 to 18 carbon atoms, as exemplified by n-capricacid, n-caprylic acid, lauric acid, myristic acid, palmitic acid,stearic acid, oleic acid and linoleic acid.

PCT Application 89/12104 (BASF Corporation) discloses a process for thepreparation of gamma and delta lactones from organic carboxylic acids orderivatives thereof by means of cultivating, under aerobic conditions, afungus of the genus Mucor in a suitable medium containing the carboxylicacid or a derivative thereof. Examples of the Mucor fungus are thefollowing strains: M. subtillissimus, M. mucedo, M. miehei, M.circinelloides, M. luteus, M. flavus, M. corticolus and M. albo-ater.Furthermore, Example 2 of PCT Application No. 89/12104 indicates thatwhen using Mucor circinelloides and using an ethyl octanoate substrate,the resulting product recovered is 49.4% gamma octalactone having thestructure: ##STR7## having a specific rotation (alpha_(d) of -39.8).

Kutney, et al, Helvetica Chimica Acta., Volume 65, Fasc. 5 (1982) No.127, at pages 1,343-1,350, discloses the use of Mortierella isabellinain carrying out a gamma hydroxylation of the compound having thestructure: ##STR8## to form the compound having the structure: ##STR9##according to the reaction: ##STR10##

U.S. Pat. No. 4,960,597 issued on Oct. 2, 1990 described a process forthe preparation of compositions of matter containing both saturated andunsaturated lactones including the saturated gamma decalactone. Thus,U.S. Pat. No. 4,960,597 described a process for the preparation ofcompositions of matter defined according to the generic structure:##STR11## wherein R represents C₆ alkyl or alkenyl, and X represents C₂,C₄ or C₆ alkylene or alkyenylene; with the proviso that R is C₆ alkylwhen X is alkyenylene and R is C₆ alkenyl when X is alkyenlene by meansof the sequential steps of (i) fermentation of castor oil or ricinoleicacid using a microorganism selected from the group consisting of:

Candida petrophilum, ATCC 20226;

Candida oleophila, ATCC 20177;

Candida sp., ATCC 20504; and

Candida sake, ATCC 28137

whereby gamma hydroxydecanoic acid and a mixture of other acids definedaccording to the generic structure: ##STR12## is formed wherein Yrepresents an oxo-saturated, oxo-unsaturated or di-unsaturated C₉, C₁₁or C₁₃ moiety according to the reaction: ##STR13## (ii) lactonization ofthe resulting gamma hydroxydecanoic acid by means of simultaneousacidification and heating according to the reaction: ##STR14## and (iii)lactonization (via distillation) of one or more of the resulting acidsdefined according to the structure: ##STR15## according to the reaction:##STR16## wherein the sum of the number of carbon atoms in the X moietyand in the R moiety is equal to the number of carbon atoms in the Ymoiety minus 1.

In the flavor and fragrance art, a need has arisen for the developmentand efficient production of naturally occurring lactones which haveheretofore been found to be useful and necessary in the creation offlavor formulations used in augmenting or enhancing the aroma or tasteof foodstuffs, chewing gums, toothpastes, medicinal products, chewingtobaccos and smoking tobaccos and also useful in augmenting or enhancingthe aroma of perfume compositions, colognes and perfumed articles (e.g.,solid or liquid anionic, cationic, nonionic or zwitterionic detergents,perfumed polymers, fabric softener compositions, fabric softenerarticles, hair preparations, cosmetic powders and the like).

Gamma octalactone defined according to the structure: ##STR17##particularly its optical isomers having the structures: ##STR18## isuseful particularly for forming butter flavors for use in flavoringproducts which cannot contain any natural butter due to health reasons.Thus, compounds having the structures: ##STR19## are found to be highlyuseful in producing butter flavored margarine. Furthermore, thecombination of the compounds having the structures: ##STR20## takenfurther with other naturally occurring materials produced viafermentation including but not limited to, for example, the compoundshaving the structures: ##STR21## has been found to be highly useful inthe production of butter flavors as well as in the production ofperfumery materials, colognes and perfumed articles.

Nothing in the prior art however discloses the ability by means offermentation to create novel mixtures of lactones together with otherfuran derivatives for use in augmenting or enhancing the organolepticproperties of consumable materials.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a block flow schematic diagram setting forth apparatus used incarrying out a process for the production of the novel compositions ofmatter of our invention including the lactone having the structure:##STR22##

FIG. 2 is a GLC profile for the reaction product of Example I (a)containing the compound having the structure: ##STR23## (Conditions:OV-1 column (50 meters×0.31 mm programmed from 75°-225° C. at 2.0° C.per minute).

FIG. 3 is the GLC profile for the reaction product of Example I (b)containing the compound having the structure; ##STR24## (Conditions: 50m×0.31 mm OV-1 column programmed from 75°-225° C. at 2.0° C. perminute).

FIG. 4 is the GLC profile for the reaction product of Example I (c)containing the compound having the structure: ##STR25## (Conditions: 50m×0.31 mm OV-1 column programmed from 75°-225° C. at 2.0° C. perminute).

FIG. 5 is the GLC profile for the reaction product of Example I (d)containing the compound having the structure: ##STR26## (Conditions: 50m×0.31 mm OV-1 column programmed from 75°-225° C. at 2.0° C. perminute).

FIG. 6 is the GLC profile for the reaction product of Example I (e)containing the compound having the structure: ##STR27## (Conditions: 50m×0.31 mm OV-1 column programmed from 75°-225° C. at 2.0° C. perminute).

FIG. 7 is the GLC profile for the reaction product of Example I (f)containing the compound having the structure: ##STR28## (Conditions: 50m×0.31 mm OV-1 column programmed from 75°-225° C. at 2.0° C. perminute).

FIG. 8 is the GLC profile for the reaction product of Example I (g)containing the compound having the structure: ##STR29## (Conditions: 50m×0.31 mm OV-1 column programmed from 75°-225° C. at 2.0° C. perminute).

FIG. 9 is the GLC profile for the reaction product of Example IIIcontaining the compound having the structure: ##STR30##

FIG. 10 is the GC mass spectrum for the combined product of Examples IIand III containing the compound having the structure: ##STR31##

FIG. 11 shows in detail the peaks indicated by reference numerals 252and 254 of the GC mass spectrum of FIG. 10.

FIG. 12 is a mass spectrum for the combined product of Examples II andIII.

FIG. 12a is a detailed section of the mass spectrum of FIG. 12.

FIG. 13 is a mass spectrum of the combined product of Examples II andIII.

FIG. 13a is a detailed section of the mass spectrum of FIG. 13.

FIG. 14 is the GLC profile for the first and second extraction of thereaction product of Example IV containing the compound having thestructure: ##STR32## (Conditions: 50 m×0.31 mm OV-1 column programmedfrom 75°-225° C. at 2.0° C. per minute).

FIG. 15 is the GLC profile for the third extraction of the reactionproduct of Example IV containing the compound having the structure:##STR33## (Conditions: 50 m×0.31 mm OV-1 column programmed at 200° C.isothermal; and from 200°-250° C. at 10° C. per minute after a tenminute period).

FIG. 16(a) is the GLC profile for distillation Fraction 2 of thereaction product of Example VI.

FIG. 16(b) is another GLC profile for distillation Fraction 2 of thereaction product of Example VI.

FIG. 17 is the GC mass spectrum for distillation Fraction 2 of ExampleVI containing the compound having the structure: ##STR34##

FIG. 18 is the mass spectrum for distillation Fraction 2 of Example VI.

FIG. 19 is the GLC profile for distillation Fraction 4 of the reactionproduct of Example VI.

FIG. 20 is the GC mass spectrum of distillation Fraction 4 of thereaction product of Example VI containing the compound having thestructure: ##STR35##

FIG. 21 is the GLC profile for distillation Fraction 5 of the reactionproduct of Example VI.

FIG. 22 is the GC mass spectrum of distillation Fraction 5 of thereaction product of Example VI containing the compound having thestructure: ##STR36##

FIG. 23 is a detailed section of the peak indicated by reference numeral284 of the GC mass spectrum of FIG. 22.

FIG. 24 is the GLC profile for the reaction product of Example VII(a)containing the compound having the structure: ##STR37##

FIG. 25 is the GLC profile for the reaction product of Example VII(b)containing the compound having the structure: ##STR38##

FIG. 26 is the GLC profile for the reaction product of Example VII(c)containing the compound having the structure: ##STR39##

FIG. 27 represents a cut-away side elevation view of apparatus usedinforming perfumed polymers which contain imbedded therein at least oneof the lactone-containing compositions of our invention.

FIG. 28 is a front view of the apparatus of FIG. 27 looking in thedirection of the arrows.

SUMMARY OF THE INVENTION

The present invention is directed to a microbiological process for theproduction of compositions containing a major proportion of gammaoctalactone generically defined according to the structure: ##STR40##containing the stereoisomers having the structures: ##STR41##

According to this process, a culture of one of the following fungi isincubated with a substrate, caprylic acid or ethyl caprylate definedaccording to the generic structure: ##STR42## wherein R is ethyl orhydrogen to form the gamma hydroxy acid defined according to thestructure: ##STR43## using a microorganism selected from the group conssting of: Mortierella ramanniana var. ramanniana--CBS 112.08

Mortierella ramanniana var. ramanniana--CBS 478.63

Choanephora cucurbitarum--NRRL 2744

Mortierella isabellina--IFO 8183

Mortierella isabellina--IFO 8309

Mortierella nana--IFO 8794

Mortierella isabellina--IFO 7824

Mortierella vinacea--IFO 6738

according to the reaction: ##STR44##

The resulting carboxylic acid is then heated in the presence of acid (asby distillation and/or evaporation) to yield the desired lactoneaccording to the reaction: ##STR45##

The resulting acid is usually an optical isomer defined according to oneof the structures: ##STR46## or a mixture of such isomers with eitherthe dextro or laevo rotatory isomer being the predominant isomer. Suchstereoisomers yield stereoisomers of the resulting octalactones definedaccording to the structures: ##STR47##

Along with the gamma octalactone, minor amounts of other products arealso produced when using the above-mentioned organisms, to wit, thecompounds having the structures: ##STR48##

The nutrient broth used according to the process of this inventionincludes the usual sources of nitrogen, carbohydrates, minerals andoxygen. Incubative fermentation conditions used according to the processinclude any pH, temperature, substrate concentration and substrate feedrate which will maintain the viability of the culture.

The inventive process may be conducted in a batch or continuous mode ofoperation. In a batch fermentation, the nutrient broth, culture andsubstrate are combined and fermented until the lactone concentrationbecomes constant. In a continuous process, the substrate in the nutrientbroth may be continuously recirculated through a fermentation reactorwith the provision that substrate and product are respectively added andremoved from the recirculating broth.

In carrying out the present invention, cultivation and fermentiveincubation of the fungus are accomplished in an aqueous medium in thepresence of the usual nutrient substances. A suitable medium is onewhich contains carbon sources, nitrogen sources, inorganic salts andgrowth factors. Among the suitable carbon sources are, for example,glucose, fructose, xylose, sucrose, maltose, lactose, mannitol,sorbitol, glycerol, corn syrup and corn syrup solids. Examples ofsuitable nitrogen sources include organic and inorganicnitrogen-containing substances such as peptone, corn steep liquor, meatextract, yeast extract, casein, urea, amino acids, ammonium salts,nitrates, enzymatic digest of soy, and mixtures thereof.

Examples of inorganic salts include the phosphate and sulfate salts ofmagnesium, sodium, calcium and potassium.

These nutrients may be supplemented with, for example, one or morevitamins of the "B" group and one or more trace minerals such as iron,manganese, cobalt and copper as desired.

For the nutrient broth, it is preferred to utilize dextrose at aconcentration of from about 2 up to about 20 weight percent, preferablyat about 10 weight percent. It is also preferred to employ "B" vitaminseither as a separate supplement or in the form of a yeast extract. Thekind and amounts of the above-mentioned additives can be determined byapplying the general knowledge in the art for the cultivation ofmicroorganisms.

In a typical procedure, one of the fungi as set forth below:

Mortierella ramanniana var. ramanniana--CBS 112.08

Mortierella ramanniana var. ramanniana--CBS 478.63

Choanephora cucurbitarum--NRRL 2744

Mortierella isabellina--IFO 8183

Mortierella isabellina--IFO 8309

Mortierella nana--IFO 8794

Mortierella isabellina--IFO 7824

Mortierella vinacea--IFO 6738

is first cultivated in inoculum quantities to produce a mature culturein nutrient broth. The culture is inoculated into a fermentor nutrientbroth and allowed to establish itself. The substrate is then added andfermentation continued until a steady concentration of lactone ispresent.

The cultivation and fermentative incubation of the fungus can be carriedout as a stationary culture or as a submerged culture (e.g.,shake-flask, fermentor), preferably under aerobic conditions.Cultivation and incubation may proceed in a pH range of from about 3 upto about 9, preferably in the range of about 5 to about 7. The pH may beregulated by the addition of an inorganic or organic acid or base suchas hydrochloric acid, acetic acid, sodium hydroxide, calcium carbonate,ammonia, ion-exchange resins, or by the addition of a buffer such as aphosphate or phthalate. The incubation temperature is suitablymaintained at between about 18° C. up to about 31° C., with a range offrom about 26°-28° C. being preferred.

In accordance with another typical procedure of the present invention,the process is conveniently carried out by adding the substrate to theculture medium at the onset of cultivation, under aerobic conditions.Alternatively, the substrate may be added either alone or in combinationwith another carbon source, such as glucose, during fermentativeincubation, or when cultivation is complete. It is preferable to add thesubstrate to the culture medium during the period of from 4 up to 24hours after the growth of the culture in the fermentative broth hascommenced. Desirable results can be obtained when the substrate is addedcontinuously over the entire fermentation after an initial fungalcultivation period of from 3 up to 12 hours. A preferred feed rate forthis continuous addition is from about 0.01 up to 1 gram per hour perliter with a preferred range of from 0.6 up to 0.8 gram per hour perliter. The concentration of the substrate in the medium may varydepending on the conditions employed. In practice, the concentration ofthe substrate in the medium may conveniently vary from 0.01% up to about10% with a preferably concentration of about 1% by weight, consistentwith the manner in which it is added to the culture.

Under the usual conditions, mixtures of optically active lactones havingthe structures: ##STR49## are produced.

Depending on the pH, oxygen flow rate, nutrients and whether R is ethylor hydrogen, the concentrations of side products will vary, these sideproducts being the compounds having the structures: ##STR50##

The reaction period for carrying out the reaction, to wit: ##STR51##varies according to the specific incubation parameters, such as thestrain of microorganism employed, the composition of the culture mediumand whether the substrate used is the ethyl caprylate ester having thestructure: ##STR52## or caprylic acid having the structure: ##STR53## Ingeneral, shake flask cultures require between 100 and 200 hours,preferably between 120 and 150 hours, depending upon the microbialstrain and the substrate utilized. However, when a fermentor is used,the fermentation period may be reduced to 40-50 hours.

The incubation is carried out under aerobic conditions, wherein thedissolved oxygen content in the incubation broth is from 20 to 100% ofsaturation by weight, preferably 30% to 80%. Also, preferably, thesubstrate is maintained in continuous contact with the aqueous phase andthe microorganism. Generally, vigorous stirring or shaking issatisfactory, but if desired a surface active agent, such as TWEEN®80,can be added to aid in the dispersion of the substrate. Conventionalantifoam agents such as silicone oils, polyalkylene glycol derivatives,or soya oil can be used to control foaming.

The form in which the microorganisms are used for the fermentation isnot critical. The fermentation may be carried out using the cells of themicroorganism isolated from the culture solution, or with an enzymeextract isolated from the cells in a known manner. In the latter case,the reaction can be conveniently carried out in an aqueous solution, forexample, in a buffer solution, in a physiological saline solution, in afresh nutrient solution, or in water. The isolated cells or an enzymeextract thereof may be immobilized on a solid support and the desiredtransformation conducted separately. It will be convenient to employ theimmobilized form of the enzyme extract in a continuous process. Thefermentation of the substrate may also be effected by mutants of thefungus.

The progress of the fermentative production of the hydroxycarboxylicacid or hydroxy carboxylic acid ester having one or both of thestructures: ##STR54## can be monitored by assaying for hydroxy acid orhydroxy ester concentration using standard analytical techniques such aschromatography (gas-liquid, thin layer or high pressure liquid) andspectroscopy such as IR and NMR. The fermentation can also be followedby measuring consumption of substrate, glucose, oxygen or by measuringpH changes. The fermentation is generally terminated when all of thesubstrate has been consumed or when no further increase in the hydroxyacid or hydroxy ester concentration is observed.

When the resulting material is sterilized as by heating, the hydroxyacid or hydroxy ester is formed into one or both of the lactones definedaccording to the structures: ##STR55## Necessarily, the reaction itselfwill cause in situ formation of lactones in a lower concentration.Resultant sterilization and subsequent distillation gives rise tocomplete conversion of the hydroxy acid and hydroxy ester to thelactones having the structures: ##STR56##

The present invention produces unexpectedly high yields of theoctalactones defined according to the structures: ##STR57## comparedwith prior art method which yield products having a much lower yield.

Hereinafter, the term "lactone derivative(s)" will be understood to meanthe reaction products subsequently purified containing substantially allof the compounds having the structures: ##STR58## together with one ormore of the side products having the structures: ##STR59##

The lactone derivative(s) and one or more auxiliary perfume ingredients,including, for example, hydrocarbons, alcohols, ketones, aldehydes,nitriles, esters other than the lactone derivatives of our invention,ethers, synthetic essential oils, and natural essential oils may beadmixed so that the combined odors of the individual components producea pleasant and desired fragrance, particularly and preferably in thefruity area (e.g., peach and apricot aromas). Such perfume compositionsusually contain (a) the main note or the "bouquet" or foundation stoneof the composition; (b) modifiers which round off and accompany the mainnote; (c) fixatives which include odorous substances which lend aparticular note to the perfume throughout all stages of evaporation andsubstances which retard evaporation; and (d) topnotes which are usuaallylow-boiling, fresh-smelling materials.

In perfume compositions, it is the individual compositions whichcontribute to their particular olfactory characteristics, however, theoverall sensory effect of the perfume composition will be at least thesum total of the effects of each of the ingredients. Thus, one or moreof the lactone derivative(s) of our invention can be used to alter,modify or enhance the aroma characteristics of a perfume composition,for example, by utilizing or moderating the olfactory reactioncontributed by another ingredient in the composition.

The amount of lactone derivative(s) of our invention which will beeffective in perfume compositions as well as in perfumed articles andcolognes depends upon many factors including the other ingredients,their amounts and the side effects which are desired. It has been foundthat perfume compositions containing as little as 0.005% of lactonederivative(s) or even less (e.g., 0.002%) can be used to impart sweet,fruity (peach and apricot) aromas to soaps, cosmetics, detergentsincluding anionic, cationic, nonionic and zwitterionic solid or liquiddetergents, perfumed polymers and other products. The amount employedcan range up to 70% of the fragrance components and will depend upon theconsideration of cost, nature of the end product, the effect desired onthe finished product and the particular fragrance sought.

The lactone derivative(s) of our invention are useful (taken alone ortaken together with other ingredients in perfume compositions) indetergents, soaps, space odorants and deodorants, perfumes, colognes,toilet waters, bath preparations, hair preparations such as lacquers,brilliantines, pomades and shampoos; cosmetic preparations such ascreams, deodorants, hand lotions and sun screens; powders such as talcs,dusting powders, face powders and the like.

As little as 0.25% of the lactone derivative(s) will suffice to impartan intense, sweet, fruity (peach and apricot) aroma to floral perfumeformulations. Generally no more than 5% of the lactone derivative(s)based on the ultimate end product is required to be used in the perfumecompositions.

Furthermore, as little as 0.25% of the lactone derivative(s) willsuffice to impart such aromas to perfumed articles per se, whether inthe presence of other perfume materials or whether used by themselves.Thus, the range of use of the lactone derivative(s) of our invention inperfumed articles, e.g., perfumed polymers and solid or liquid anionic,cationic, nonionic or zwitterionic solid or liquid detergents, may varyfrom 0.25% up to about 5% by weight based on the total weight of theperfumed article.

In addition the perfume composition or fragrance composition of ourinvention can contain a vehicle or carrier for the lactonederivative(s). The vehicle can be a liquid such as a non-toxic alcohol,e.g., ethanol, a non-toxic glycol, e.g., propylene glycol, or the like.The carrier can also be an absorbent solid such as a gum (e.g., gumarabic or xanthan gum or guar gum) or components for encapsulating thecomposition by means of coacervation (such as by gelatin) or by means offormation of a polymer around a liquid center (as by using a ureaformaldehyde prepolymer to form a polymeric capsule around a perfumecomposition center).

It will be appreciated from the present disclosure that the lactonederivative(s) according to the present invention can be used to alter,vary, fortify, modify, enhance or otherwise improve the flavor of a widevariety of materials which are ingested, consumed or otherwiseorganoleptically sensed.

The terms "alter" and "modify" in their various forms will be understoodherein to mean the supplying or imparting of a flavor character or noteto an otherwise bland, relatively tasteless substance, or augmenting anexisting flavor characteristic where the natural flavor is deficient insome regard or supplementing the existing flavor impression to modifyits organoleptic character.

The term "enhance" is intended herein to mean the intensification (byuse of the lactone derivative of our invention) of a flavor or aromanote or nuance in a tobacco flavor or foodstuff or perfume compositionor perfumed article without changing the quality of said note or nuance.

A "flavoring composition" is taken to mean one which contributes a partof the overall flavor impression by supplementing or fortifying anatural or artificial flavor in a material or one which suppliessubstantially all the flavor and/or aroma character to a consumablearticle.

The term "foodstuff" as used herein includes both solid and liquidingestible materials for man or animals which materials usually do, butneed not, have nutritional value. Thus, foodstuffs include meats,gravies, soups, convenience foods, malt, alcoholic and other beverages,milk and dairy products, seafoods, including fish, crustaceans, mollusksand the like, candies, vegetables, cereals, soft drinks, snacks, dog andcat foods, other veterinary products, and the like. The lactonederivative(s) of our invention are also useful in tobacco flavorants andflavor enhancers.

The term "tobacco" will be understood herein to mean natural productssuch as, for example, burley, Turkish tobacco, Maryland tobacco,flue-cured tobacco and the like including tobacco-like or tobacco-basedproducts such as reconstituted or homogenized leaf and the like as wellas toacco substitutes intended to replace natural tobacco such aslettuce and cabbage leaves and the like. The tobaccos and tobaccoproducts in which the lactone derivative(s) of our invention are usefulincluding those designed or used for smoking such as in cigarettes,cigar and pipe tobacco, as well as products such as snuff, chewingtobacco and the like.

When the lactone derivative(s) of this invention are used in a flavoringcomposition, they can be combined with conventional flavoring materialsor adjuvants. Such co-ingredients or flavor adjuvants are well known inthe art for such and have been extensively described in the literature.Requirements of such adjuvant materials are: (1) that they benon-reactive with the lactone derivative(s) of our invention; (2) thatthey be organoleptically compatible with the lacton derivative(s) of ourinvention whereby the flavor of the ultimate consumable material towhich the lactone derivative(s) are added is not detrimentally affectedby the use of the adjuvant; (3) that they be ingestibly acceptable andthus non-toxic or otherwise non-deleterious. Apart from theserequirements, conventional materials can be used and broadly includeother flavor materials, vehicles, stabilizers, thickeners, surfaceactive agents, conditioners, and flavor intensifers.

Such conventional flavoring materials include saturated fatty acids,unsaturated fatty acids and amino acids; alcohols including primary andsecondary alcohols, esters, carbonyl compounds including ketones andaldehydes; lactones; other cyclic organic materials including benzenederivates, allicyclic compounds, heterocyclics such as furans,pyridines, pyrazines and the like; sulfur-containing compounds includingthiols, sulfides, disulfides and the like; proteins; lipids,carbohydrates; so-called flavor potentiators such as monosodiumglutamate; magnesium glutamate, calcium glutamate, guanylates andinosinates; natural flavoring materials such as cocoa, vanilla andcaramel; essential oils and extracts such as anise oil, clove oil andthe like and artificial flavoring materials such as vanillin and thelike.

Specific preferred flavor adjuvants are as follows:

anise oil;

ethyl-2-methyl butyrate;

vanillin;

cis-3-heptenol;

cis-3-hexenol;

trans-2-heptenol;

cis-3-heptenal;

butyl valerate;

2,3-diethyl pyrazine;

methyl cyclopentenolone;

benzaldehyde;

valerian oil;

3,4-dimethoxyphenol;

amyl acetate;

amyl cinnamate;

gamma butyryl lactone;

furfural;

trimethyl pyrazine;

phenyl acetic acid;

isovaleraldehyde;

ethyl maltol;

ethyl vanillin;

ethyl valerate;

ethyl butyrate;

cocoa extract;

coffee extract;

peppermint oil;

spearmint oil;

clove oil;

anethol;

cardamom oil;

wintergreen oil;

cinnamic aldehyde;

ethyl-2-methyl valerate;

gamma hexenyl lactone;

2,4-decadienal;

2,4-heptadienal; and

According to another aspect of our invention, an organolepticallyimproved smoking tobacco product and additives therefor as well asmethods of making the same which overcome specific problems heretoforeencountered in which specific Turkish, oriental-like aromas prior tosmoking and improved Turkish, oriental aromas on smoking in the mainstream and the side stream are created or enhanced or modified oraugmented and may be readily controlled and maintained at the desireduniform level regardless of variations in the tobacco components of theblend. In particular, low grade Virginia-type tobaccos may be upgradedusing the lactone derivative(s) of our invention.

This invention further provides improved tobacco additives and methodswhereby various desirable natural aromatic Turkish tobacco flavoringcharacteristics with oriental notes may be imparted to smoking tobaccoproducts and may be readily varied and controlled to produce the desireduniform flavoring characteristics.

In carrying out this aspect of our invention, we add to smoking tobaccomaterials or a suitable substitute therefor (e.g., dried lettuce leaves)an aroma and flavor additive containing as an active ingredient one ormore of the lactone derivative(s) of our invention.

In addition to the lactone derivative(s) of our invention, otherflavoring and aroma additives may be added to the smoking tobaccomaterial or substitute therefor either separately or in admixture withthe lactone derivative(s) of our invention as follows:

I. Synthetic Materials

Beta-ethyl-cinnamaldehyde;

Eugenol;

Dipentene;

Beta-damascenone;

Maltol;

Ethyl maltol;

Delta undecalactone;

Delta decalactone;

Benzaldehyde;

Amyl acetate;

Ethyl butyrate;

Ethyl valerate;

Ethyl acetate;

2-Hexenol-1;

2-Methyl-5-isopropyl-1,3-nonadiene-8-one;

2,6-Dimethyl-1,6-undecadiene-10-one;

2-Methyl-5-isopropyl acetophenone;

2-Hydroxy-2,5,5,8a-tetramethyl-1-(2-hydroxyethyl)decahydronaphthalene;

Dodecahydro-3a,6,6,9a-tetramethyl naphtho(2,1-b) furan;

4-Hydroxy hexanoic acid, gamma lactone; and

Polyisoprenoid hydrocarbons defined in Example V of U.S. Pat. No.3,589,372 issued on Jun. 29, 1971.

II. Natural Oils

Celery seed oil;

Coffee extract;

Bergamot oil;

Cocoa extract;;

Nutmeg oil; and

Origanum oil.

An aroma and flavoring concentrate containing one or more of the lactonederivative(s) of our invention and, if desired, one or more of the aboveindicated additional flavoring additives may be added to the smokingtobacco material, to the filter or to the leaf or paper wrapper. Thesmoking tobacco material may be shredded, cured, cased and blendedtobacco material or reconstituted tobacco material or tobaccosubstitutes (e.g., lettuce leaves) or mixtures thereof. The proportionsof flavoring additives may be varied in accordance with taste butinsofar as enhancement or the imparting of oriental and/or Turkishtobacco notes, we have found that satisfactory results are obtained ifthe proportion by weight of the sum total of lactone derivative(s) tosmoking tobacco material is between 50 ppm and 1,500 ppm (0.005%-0.15%)of the active ingredients to the smoking tobacco material. We havefurther found that satisfactory results are obtained if the proportionby weight of the sum total of lactone derivative(s) used to flavoringmaterial is between 500 and 15,000 ppm (0.05%-1.5%).

Any convenient method for incorporating the lactone derivative(s) intothe tobacco product may be employed. Thus, the lactone derivative(s)taken alone or along with other flavoring additives may be dissolved ina suitable solvent such as ethanol, diethylether, and/or volatileorganic solvents and the resulting solution may either be spread ontothe cured, cased, and blended tobacco material or the tobacco materialmay be dipped into such solution. Under certain circumstances, asolution of the lactone derivative(s) taken alone or taken furthertogether flavoring additives as set forth above may be applied by meansof a suitable applicator such as a brush or roller on the paper or leafwrapper for the smoking product, or it may be applied to the filter byeither spraying or dipping or coating.

Furthermore, it will be apparent that only a portion of the tobacco orsubstitute therefore need be treated and the thus-treated tobacco may beblended with other tobaccos before the ultimate tobacco product isformed. In such cases, the tobacco treated may have the lactonederivative(s) in excess of the amounts or concentrations above indicatedso that when blended with other tobaccos, the final product will havethe percentage within the indicated range.

In accordance with one specific example of our invention, an aged, curedand shredded domestic Virginia tobacco is sprayed with a 20% alcoholsolution of the compound having the structure: ##STR60## on a dry basis.Thereafter, the alcohol is removed by evaporation and the tobacco ismanufactured into cigarettes by the usual techniques. The cigarette,when treated as indicated, has a desired and pleasing aroma which isdetectable in the main stream and the side stream when the cigarette issmoked. The aroma is described as being sweeter, with pronouncedTurkish/oriental characteristics and with improved body and enhancedtobacco character in the main stream and side stream. In addition,interesting amber nuances are imparted.

While our invention is particularly useful in the manufacture of smokingtobacco such as cigarette tobacco, cigar tobacco and pipe tobacco, othertobacco products formed from sheeted tobacco dust or fines may also beused. Likewise the lactone derivative(s) of our invention can beincorporated with materials such as filter tip materials, seam paste,packaging materials and the like which are used along with tobacco toform a product adapted for smoking. Furthermore, the lactonederivative(s) can be added to certain tobacco substitutes of natural orsynthetic origin (e.g., dried lettuce leaves) and, accordingly, by theterm "tobacco" as used throughout this specification, is meant anycomposition intended for human consumption by smoking or otherwise whencomposed of tobacco plant parts or substitute material or both.

The lactone derivative(s) of our invention can be used to alter, vary,fortify, modify, enhance or otherwise improve the organolepticproperties, including flavor and/or aroma, of a wide variety ofmaterials which are ingested, consumed, or otherwise organolepticallysensed.

The term "alter" in its various forms will be understood herein to meanthe supplying or imparting of a flavor character or note to an otherwisebland, relatively tasteless substance, or augmenting the existing flavorcharacteristic where the natural flavor is deficient in some regard, orsupplementing the existing flavor or aroma impression to modify theorganoleptic character. The materials which are so altered are generallyreferred to herein as consumable materials.

The lactone derivative(s) of our invention are accordingly useful inflavoring compositions. Flavoring compositions are hereinafter taken tomean those which contribute a part of the overall flavor impression bysupplementing or fortifying a natural or artificial flavor in amaterial, as well as those which supply substantially all the flavorand/or aroma character to a consumable article.

The term "foodstuff" as used herein includes both solid and liquidingestible materials for man or animals, which materials usually do, butneed not, have nutritional value. Thus, foodstuffs include meats,gravies, soups, convenience foods, malt and other alcoholic ornon-alcoholic beverages, milk and dairy products, (including but notlimited to margarine and butter) nut butters such as peanut butter andother spreads, seafoods including fish, crustaceans, mollusks and thelike, candies, breakfast foods, baked goods, vegetables, cereals, softdrinks, snack foods, dog and cat foods, other veterinary products, andthe like.

When the lactone derivative(s) produced according to this invention areused in a food flavoring composition, they can be combined withconventional flavoring materials or adjuvants. Such co-ingredients orflavoring adjuvants are well known in the art for such use and have beenextensively described in the literature. Apart from the requirement thatany such adjuvant material is ingestibly acceptable, and thus non-toxicor otherwise non-deleterious, conventional materials can be used andbroadly include other flavor materials, vehicles, stabilizers,thickeners, surface active agents, conditioners and flavor intensifiers.

Examples of preferred co-flavoring adjuvants are:

Methyl thiazole alcohol (4-methyl-5-beta-hydroxyethyl thiazole);

2-Methyl butanethiol;

4-Mercapto-2-butanone;

3-Mercapto-2-pentanone;

1-Mercapto-2-propanone;

Benzaldehyde;

Furfural;

Furfuryl alcohol;

2-Mercapto propionic acid;

Alkyl pyrazine;

Methyl pyrazine;

2-Ethyl-3-methyl pyrazine;

Tetramethyl pyrazine;

Polysulfides;

Dipropyl disulfide;

Methyl benzyl disulfide;

Alkyl thiophenes;

2-Butyl thiophene;

2,3-Dimethyl thiophene;

5-Methyl furfural;

Acetyl furan;

2,4-Decadienal;

Guiacol;

Phenyl acetaldehyde;

β-Decalactone;

d-Limonene;

Acetoin;

Amyl acetate;

Maltol;

Ethyl butyrate;

Levulinic acid;

Piperonal;

Ethyl acetate;

n-Octanal;

n-Pentanal;

n-Hexanal;

Diacetyl;

Monosodium glutamate;

Monopotassium glutamate;

Sulfur-containing amino acids, e.g., Cysteine;

Hydrolyzed vegetable protein;

2-Methylfuran-3-thiol;

2-Methyldihydrofuran-3-thiol;

2,5-Dimethylfuran-3-thiol;

Hydrolyzed fish protein;

Tetramethyl pyrazine;

Propylpropenyl disulfide;

Propylpropenyl trisulfide;

Diallyl disulfide;

Diallyl trisulfide;

Dipropenyl disulfide;

Dipropenyl trisulfide;

4-Methyl-2-[methylthio)-ethyl]-1,3-dithiolane;

4,5-Dimethyl-2-[methylthio)ethyl]-1,3-dithiolane;

4,5-Dimethyl-2-(methylthiomethyl)-1,3-dithiolane; and

4-Methyl-2-(methylthiomethyl)-1,3-dithiolane.

The lactone derivative(s) of our invention or compositions incorporatingthem, as mentioned above, can be combined with one or more vehicles orcarriers for adding them to the particular product. Vehicles can beedible or otherwise suitable materials such as ethyl alcohol, propyleneglycol, water and the like. Carriers include materials such as gumarabic, carrageenan, xanthan gum, guar gum and the like.

The lactone derivative(s) prepared according to this invention can beincorporated with the carriers by conventional means such asspray-drying, drum-drying and the like. Such carriers can also includematerials for coacervating the lactone derivative(s) of our invention toprovide encapsulated products. When the carrier is an emulsion theflavoring composition can also contain emulsifiers such as mono- anddiglycerides or fatty acids and the like. With these carriers orvehicles, the desired physical form of the composition can be prepared.

The quantity of lactone derivative(s) utilized should be sufficient toimpart the desired flavor characteristic to the product, but on theother hand, the use of an excessive amount of the lactone derivative(s)is not only wasteful and uneconomical, but in some instances too large aquantity may unbalance the flavor or other organoleptic properties ofthe product consumed. The quantity used will vary depending upon theultimate foodstuff; the amount and type of flavor initially present inthe foodstuff; the further process or treatment steps to which thefoodstuff will be subjected; regional and other preference factors; thetype of storage if any to which the product will be subject; and thepreconsumption treatment, such as baking, frying, and so on, given tothe product by the ultimate consumer. Accordingly, the terminology"effective amount" and "sufficient amount" is understood in the contextof the present invention to be quantitatively adequate to alter theflavor of the foodstuff.

It is accordingly preferred that the ultimate composition contain fromabout 0.1 parts per million (ppm) to about 500 ppm of the lactonederivative(s).

The lactone derivative(s) of our invention when utilized in flavoringcompositions can be varied over a wide range depending upon theparticular flavor nuances desired to be added to the foodstuff. Thusamounts of the lactone derivative(s) of our invention may be containedin flavoring materials from about 1 ppm up to about 50% by weight of theflavoring composition. Indeed, the compounds having the structures:##STR61## as well as the hydroxy acids having the structures: ##STR62##may be utilized in margarine flavors at levels of between about 1% andabout 50%.

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1 is the block flow diagram setting forth in schematic form theapparatus used in carrying out the process for producing the lactonederivative(s) of our invention. Into fermentor 10, medium 12 is passedthrough line 14 using pump 16 and control valve 18. The addition of themedium into the fermentor 10 is followed by the addition of preparedculture from location 26 through line 32 past pump 28 using controlvalve 30. The addition of the prepared culture to the medium is followedby the addition of the substrate from location 20 through line 22 usingcontrol valve 24.

The fermentation is carried out for a period of, for example, 48 hours.

The fermentation broth is then passed through line 34 using pump 36 pastcontrol valve 38 through line 42 into extractor 40 wherein extractantfrom location 44 and from feed tank 52 is passed through line 54 usingpump 58 past control valve 56 into the extractor 40. The extractiontakes place in extractor 40 and the lactone-containing extractionsolvent is evaporated in evaportor 68 after being fed into theevaporator through line 64 using control valve 66. The extractionsolvent from location 44 is passed through line 50 using pump 48 pastcontrol valve 46 into feed tank 52. Solvent evaporated from theevaporator 68 passes through line 70 past control valve 72 into the feedtank 52 recycled and original extraction solvent from feed tank 52passes through line 54 using pump 58 past control valve 56 into theextractor 40.

The resulting product containing a high proportion of gamma octalactonehaving the structure: ##STR63## is passed through line 74 intodistillation column 76 wherein the octalactone is distilled overheadusing reflux head 88 having therein control valve 86 and 92 and recycleline 90. The octalactone passes through line 94 into holding tank 96where it may further be distilled or extracted and used for itsorganoleptic properties. The bottoms from distillation column 76 passesthrough line 78 using control valve 80 into reboiler 98 where reboiledproduct passes through line 84 using control valve 82. The bottoms fromthe distillation column 76 are held in holding tank 99.

FIG. 2 is the GLC profile for the reaction product of Example I(a). Thepeak indicated by reference numeral 200 is the peak for methyl caprylatehaving the structure: ##STR64## The peak indicated by reference numeral202 is the peak for ethyl caprylate having the structure: ##STR65## Thepeak indicated by reference numeral 204 is the peak for gammaoctalactone having the structure: ##STR66## (Conditions: 50 m×0.31 mmOV-1 column programmed from 75°-225° C. at 2.0° C. per minute).

FIG. 3 is the GLC profile for the reaction product of Example I(b). Thepeak indicated by reference numeral 206 is the peak for methyl caprylatehaving the structure: ##STR67## The peak indicated by reference numeral208 is the peak for ethyl caprylate having the structure: ##STR68## Thepeak indicated by reference numeral 210 is the peak for gammaoctalactone having the structure: ##STR69## (Conditions: 50 m×0.31 mmOV-1 column programmed from 75°-225° C. at 2.0° C. per minute).

FIG. 4 is the GLC profile for the reaction product of Example I(c). Thepeak indicated by reference numeral 214 is the peak for the ethylcaprylate having the structure: ##STR70##

The peak indicated by reference numeral 212 is the peak for methylcaprylate having the structure: ##STR71## The peak indicated byreference numeral 216 is the peak for gamma octalactone having thestructure: ##STR72## (Conditions: 50 m×0.31 mm OV-1 column programmedfrom 75°-225° C. at 2.0° C. per minute).

FIG. 5 is the GLC profile for the reaction product of Example I(d). Thepeak indicated by reference numeral 218 is the peak for methylcaprylate. The peak indicated by reference numeral 220 is the peak forethyl caprylate. The peak indicated by reference numeral 222 is the peakfor gamma octalactone. (Conditions: 50 m×0.31 mm OV-1 column programmedfrom 75°-225° C. at 2.0° C. per minute).

FIG. 6 is the GLC profile for the reaction product of Example I(e). Thepeak indicated by reference numeral 224 is the peak for methylcaprylate. The peak indicated by reference numeral 226 is the peak forethyl caprylate. The peak indicated by reference numeral 228 is the peakfor gamma octalactone. (Conditions: 50 m×0.31 mm OV-1 column programmedfrom 75°-225° C. at 2.0° C. per minute).

FIG. 7 is the GLC profile for the reaction product of Example I(f). Thepeak indicated by reference numeral 230 is the peak for methylcaprylate. The peak indicated by reference numeral 232 is the peak forethyl caprylate. The peak indicated by reference numeral 234 is the peakfor gamma octalactone. (Conditions: 50 m×0.31 mm OV-1 column programmedfrom 75°-225° C. at 2.0° C. per minute).

FIG. 8 is the GLC profile for the reaction product of Example I(g). Thepeak indicated by reference numeral 236 is the peak for methylcaprylate. The peak indicated by reference numeral 238 is the peak forethyl caprylate. The peak indicated by reference numeral 240 is the peakfor gamma octalactone. (Conditions: 50 m×0.31 mm OV-1 column programmedfrom 75°-225° C. at 2.0° C. per minute).

FIG. 9 is the GLC profile for the reaction product of Example III. Thepeak indicated by reference numeral 242 is the peak for methylcaprylate. The peak indicated by reference numeral 244 is the peak forethyl caprylate. The peak indicated by reference numeral 246 is the peakfor gamma octalactone.

FIG. 10 is the GC-mass spectrum for the reaction products of Examples IIand III, combined. The peak indicated by reference numeral 248 is thepeak for the compound having the structure: ##STR73## The peak indicatedby reference numeral 250 is the peak for the compound having thestructure: ##STR74## The peak indicated by reference numeral 252 is thepeak for caprylic acid having the structure: ##STR75## The peakindicated by reference numeral 254 is the peak for the compound havingthe structure: ##STR76## The peak indicated by reference numeral 256 isfor gamma octalactone having the structure: ##STR77## The peak indicatedby reference numeral 258 is for the compound having the structure:##STR78##

FIG. 11 is an enlargement of peaks 252 and 254 of FIG. 10. The peakindicated by reference numeral 252 is for caprylic acid having thestructure: ##STR79## The peak indicated by reference numeral 254 is forthe compound having the structure: ##STR80## hydroxy methyl furfural.

FIG. 14 is the GLC profile for the first and second extraction of thereaction product of Example IV. The peak indicated by reference numeral260 is the peak for gamma octalactone.

FIG. 15 is the GLC profile for the third extraction of the reactionproduct of Example IV. The peak indicated by reference numeral 262 iffor gamma octalactone. (Conditions: 50 m×0.31 mm OV-1 column programmedat 200° C. isothermal for 10 minutes followed by 200°-225° C. at 10° C.per minute).

FIG. 17 is the GC-mass spectrum for distillation Fraction 2 of thedistillation product of the reaction product of Example VI. The peakindicated by reference numeral 264 is the peak for the compound havingthe structure: ##STR81## The peak indicated by reference numeral 266 isfor the compound having the structure: ##STR82## The peak indicated byreference numeral 268 is the peak for the compounds having thestructures: ##STR83## The peak indicated by reference numeral 270 is thepeak for gamma octalactone having the structure: ##STR84## The peakindicated by reference numeral 272 is for the compound having thestructure: ##STR85##

FIG. 20 is the GC-mass spectrum for distillation Fraction 4 of thedistillation product of the reaction product of Example VI. The peakindicated by reference numeral 274 is the peak for the compound havingthe structure: ##STR86## The peak indicated by reference numeral 276 isthe peak for the compound having the structure: ##STR87## The peakindicated by reference numeral 278 is the peak for the compound havingthe structure: ##STR88## The peak indicated by reference numeral 280 isthe peak for gamma octalactone having the structure: ##STR89## The peakindicated by reference numeral 282 is the peak for the compound havingthe structure: ##STR90##

FIG. 22 is the GC mass spectrum for distillation Fraction 5 of thedistillation product of the reaction product of Example VI. The peakindicated by reference numeral 284 is for gamma octalactone having thestructure: ##STR91## The peak indicated by reference numeral 286 is forthe compound having the structure: ##STR92##

FIG. 23 is an enlargement of peak 284 of FIG. 22. Peak 284 is for gammaoctalactone having the structure: ##STR93##

FIG. 24 is the GLC profile for the reaction product of Example VII(a).The peak indicated by reference numeral 288 is the peak for gammaoctalactone having the structure: ##STR94##

FIG. 25 is the GLC profile for the reaction product of Example VII(b).The peak indicated by reference numeral 290 is the peak for gammaoctalactone having the structure: ##STR95##

FIG. 26 is the GLC profile for the reaction product of Example VII(c).The peak indicated by reference numeral 292 is the peak for gammaoctalactone having the structure: ##STR96##

Referring to FIGS. 27 and 28, there is provided a process for formingscented polymer elements (wherein the polymer may be a thermoplasticpolymer such as low density polyethylene or polypropylene or copolymersof ethylene and vinyl acetate or mixtures of polymers and copolymerssuch as copolymers of ethylene and vinyl acetate and polyethylene) suchas pellets useful in the formation of plastic particles useful infabricating certain articles which may be perfumed. This processcomprises heating the polymer or mixture of polymers to the meltingpoint of said polymer or mixture of polymers, e.g., 250° C. in the caseof low density polyethylene. The lower most portion of the container ismaintained at a slightly lower temperature and the material in thecontainer is taken off at such location for delivery through theconduit. Thus, referring to FIGS. 27 and 28, in particular, theapparatus used in producing such elements comprises a device for formingthe polymer containing perfume, e.g., polyethylene orpolyethylene-polyvinyl acetate of mixtures of same or polypropylene,which comprises a vat or container 1212 into which the polymer takenalone or in admixture with other copolymers and the perfuming substancewhich is at least one of the lactones of our invention or mixtures oflactones and other compatible perfumes is placed. The container isclosed by means of an air-tight lid 1228 and clamped to the container bybolts 1265. A stirrer 1273 traverses the lid or cover 1228 in anair-tight manner and is rotatable in a suitable manner. A surroundingcylinder 1212A having heating coils which are supplied with electriccurrent through cable 1214 from a rheostat or control 1216 is operatedto maintain the temperature inside the container 1212 such that thepolymer in the container will be maintained in the molten or liquidstate. It has been found advantageous to employ polymers at such atemperature that the viscosity will be in the range of 90-100 sayboldtseconds. The heater 1218 is operated to maintain the upper portion ofthe container 1212 within a temperature range of, for example, 220°-270°C. in the case of low density polyethylene. The bottom portion of thecontainer 1212 is heated by means of heating coils 1212A regulatedthrough the control 1220 connected thereto through a connecting wire1222 to maintain the lower portion of the container 1212 within atemperature range of 220°-270° C.

Thus, the polymer or mixture of polymers added to the container 1212 isheated from 10-12 hours, whereafter the perfume composition or perfumematerial which contains one or more of the lactones of our invention isquickly added to the melt. Generally, about 10-45 percent by weight ofthe resulting mixture of the perfumery substance is added to thepolymer.

After the perfume material is added to the container 1212, the mixtureis stirred for a few minutes, for example, 5-15 minutes and maintainedwithin the temperature ranges indicated previously by the heating coil1212A. The controls 1216 and 1220 are connected through cables 1224 and1226 to a suitable supply of electric current for supplying the powerfor heating purposes.

Thereafter, the valve "V" is opened permitting the mass to flowoutwardly through conduit 1232 having a multiplicity of orifices 1234adjacent to the lower side thereof. The outer end of the conduit 1232 isclosed so that the liquid polymer in intimate admixture with one or moreof the lactones of our invention or mixture of perfume substance and oneor more of the lactones of our invention, will continuously drop throughthe orifices 1234 downwardly from the conduit 1232. During this time,the temperature of the polymer intimately admixed with the perfumerysubstance in the container 1212 is accurately controlled so that atemperature in the range of from about 240°-250° C., for example, (inthe case of low density polyethylene) will exist in the conduit 1232.The regulation of the temperature through the controls 1216 and 1220 isessential in order to insure temperature balance to provide for thecontinuous dropping or dripping of molten polymer intimately admixedwith the perfume substance which is all or which contains one or more ofthe lactones of our invention, through the orifices 1234 at a rate whichwill insure the formation of droplets 1236 which will fall downwardlyonto a moving conveyor belt 1238 caused to run between conveyor wheels1240 and 1242 beneath the conduit 1232.

When the droplets 1236 fall onto the conveyor 1238, they form pellets1244 which harden almost instantaneously and fall off the end of theconveyor 1238 into a container 1250 which is advantageously filled withwater or some other suitable cooling liquid to insure the rapid coolingof each of the pellets 1244. The pellets 1244 are then collected fromthe container 1250 and utilized for the formation of other functionalproducts, e.g., garbage bags and the like.

The following examples are given to illustrate embodiments of theinvention as it is preferred to practice it. It will be understood thatthese examples are illustrative and the invention is not to beconsidered as restrictive thereto except as indicated in the appendedclaims.

All parts, proportions, percentages and ratios hereinafter referred toare by weight unless otherwise indicated.

EXAMPLE I Production of Gamma Octalactone in Shake Flasks ##STR97##

100 ml Shake flasks operating at 25° C. and 150 rpm (starting pH 4.5)are filled with medium and substrate as set forth below.

Each of the flasks is inoculated with a different organism as is shownfor Examples I(a), I(b), I(c), I(d), I(e), I(f), I(g), I(h), I(i), I(j),I(k), I(1), I(m), I(n) and I(o):

    __________________________________________________________________________    Medium:          Substrate:  Shake Flask Conditions:                          __________________________________________________________________________     1.0% Peptone    1% Ethyl Caprylate                                                                        100 ml volume                                     0.5% Yeast extract          Temperature = 25° C.                      0.05% TWEEN ® 80         Agitation = 150 rpm                              10.0% Dextrose               Starting pH = 4.5                                The results for each of the Examples are set forth below:                     __________________________________________________________________________                                      %-Octalactone                                                  Time At Which  In Reaction Product                                            Substrate Added                                                                              At End of 6 Days                                               After Commence-                                                                        Crude Wt.                                                                           After Addition                              Example                                                                            Organism      ment of Shaking                                                                        of Product                                                                          Of Substrate                                __________________________________________________________________________    I(a) Mortierella isabellina,                                                                     24 hours 0.372 grams                                                                         1.4                                              ATCC 44583                                                               I(b) Mortierella isabellina,                                                                     24 hours 0.147 grams                                                                         8.4                                              ATCC 38063                                                               I(c) Syncephalastrum racemosum,                                                                  24 hours 0.702 grams                                                                         4.1                                              NRRL A-5889                                                              I(d) Mortierella isabellina,                                                                     24 hours 0.289 grams                                                                         21.15                                            IFO 7884;                                                                I(e) Mortierella ramanniana var.                                                                 48 hours 0.465 grams                                                                         1.28                                             angulispora, IFO 8187;                                                   I(f) Mortierella isabellina,                                                                     24 hours 0.311 grams                                                                         10.15                                            CBS 221.29;                                                              I(g) Mortierella isabellina,                                                                     24 hours 0.441 grams                                                                         24.56                                            IFO 7873;                                                                1(h) Mortierella remanniana var.                                                                 24 hours 0.53 grams                                                                          37.8                                             ramanniana CBS 112.08                                                    1(i) Mortierella ramanniana var.                                                                 24 hours 0.59 grams                                                                          3.35                                             CBS 478.63                                                               1(j) Choanephora cucurbitarum                                                                    48 hours 0.66 grams                                                                          0.41                                             NRRL 2744                                                                1(k) Mortierella isabellina                                                                      24 hours 0.93 grams                                                                          1.0                                              IFO 8183                                                                 1(l) Mortierella isabellina                                                                      24 hours 0.60 grams                                                                          1.91                                             IFO 8309                                                                 1(m) Mortierella nana                                                                            48 hours 1.07 grams                                                                          1.67                                             IFO 8794                                                                 1(n) Mortierella isabellina                                                                      24 hours 0.62 grams                                                                          6.59                                             IFO 7824                                                                 1(o) Mortierella vinacea                                                                         48 hours 0.51 grams                                                                          8.37                                             IFO 6738                                                                 __________________________________________________________________________     All flasks were analyzed 6 days after addition of substrate.             

EXAMPLE II Preparation of Gamma Octalactone Using Mortierella IsabellinaIFO 7873 ##STR98##

The following medium is prepared:

1% Peptone

0.5% Yeast extract

0.05% TWEEN®80

10.0% Dextrose

Mortierella isabellina, IFO 7873 is placed into 100 ml of the abovemedium and a culture is produced. 500 ml of the above-medium is theninoculated with 2% of the above-formed culture.

A production medium is prepared as follows:

1% Peptone

0.5% Yeast extract

0.05% TWEEN®80

10.0% Dextrose.

The production medium is placed into a 9 liter fermentor equipped with a500 rpm agitator and operating at an aeration rate of 0.5 v/v/m and a pHof 4.5. The production medium is sterilized at 121° C. for 30 minutes.

The fermentor is inoculated with 0.5 liters of the prepared inoculum. 25Hours after the fermentor is inoculated, ethyl caprylate having thestructure: ##STR99## addition is commenced. The pumping of the ethylcaprylate is carried out at a rate of 0.22 grams/hour/liter. The pumpingof the ethyl caprylate is carried on for a period of 48 hours.

At the end of the fermentation the pH of the broth is adjusted tobetween 2 and 4 using 85% phosphoric acid, and then sterilized for 15minutes at 121° C. The broth is extracted 3 times with 1/3 volume ofethyl acetate. The combined extracts are washed twice with saturatedsodium chloride solution (aqueous) and the solvent is evaporated. Thecrude is analyzed as methyl esters and is then distilled on a 12"Goodloe column at a vapor temperature of 92° C. and a vacuum of 1 mm/Hg.The yield is 1.72 grams per liter of gamma octalactone. The percentoctalactone recovered is 15.64. The crude weight of the gammaoctalactone is 11 grams per liter.

EXAMPLE III Preparation of Gamma Octalactone Using MortierellaIsabellina, IFO 7873 ##STR100##

A medium is prepared containing the following ingredients:

1% Peptone

0.5% Yeast extract

0.05% TWEEN®80

10.0% Dextrose

A slant of Mortierella isabellina, IFO 7873 is placed into 100 ml of theabove medium. The inoculum is cultured for a period of 3 days. Twopercent of the thus-obtained culture is placed into 500 ml of the abovemedium and the inoculum is prepared by growing the culture in the mediumat a pH of 4.5 for a period of 24 hours.

A production medium is prepared in a 9 liter fermentor as follows:

1% Peptone

0.5% Yeast extract

0.05% TWEEN®80

10.0% Dextrose

and maintained at 27° C. with agitation of 500 rpm, aeration of 0.5v/v/m and maintained at a pH of 4.5.

The fermentor medium is sterilized at a 121° C. for 30 minutes. Thefermentor is then inoculated with 0.5 liters of the above-preparedculture. The fermentor is maintained at 27° C. and operated at 500 rpmfor a period of 5 hours after the inoculation.

At the 5 hour time interval, addition of the substrate, the compoundhaving the structure: ##STR101## is commenced at the rate of 0.25g/hr/L.

The ethyl caprylate addition is continued for a period of 48 hours.

At the end of the 48 hour period, addition of ethyl caprylate ceases.The pH of the fermentation broth is adjusted to between 2 and 3 using85% phosphoric acid. The broth is then extracted three times withone-third volume of ethyl acetate each time and the solvent isevaporated.

The crude extract is then fractionally distilled at a vapor temperatureof 92° C. and a pressure of 1 mm/Hg. yielding the product having thestructure: ##STR102##

The reaction product contained 34.72% gamma octalactone and the yieldwas 2.62 grams per liter. The crude weight of the gamma octalactone was7.56 grams per liter.

EXAMPLE IV Preparation of Gamma Octalactone Using MortierellaIsabellina, IFO 7873 ##STR103##

The following medium is prepared:

1.0% Peptone

0.5% Yeast extract

0.05% TWEEN®80

10.0% Dextrose.

To 100 ml of the above medium, a slant of Mortierella isabellina, IFO7873 is added. An inoculum is cultured for a period of 3 days. At theend of the 3 day period, the resulting culture is added to 500 ml of theabove medium at the rate of 2%. A culture is grown in the 500 ml batchfor a period of 24 hours at a pH of 4.5.

The medium:

1.0% Peptone

0.5% Yeast extract

0.05% TWEEN®80

10.0% Dextrose

is added to a 9 liter fermentor operating at 27° C., 500 rpm agitation;an aeration rate of 0.5 v/v/m and a pH of 6.5.

The above 500 ml of culture is then inoculated into the medium in thefermentor with stirring and aeration.

Five hours after inoculation, pumping of ethyl caprylate having thestructure: ##STR104## is commenced into fermentor at the rate of 0.25g/hr/L. The pumping of the ethyl caprylate is continued over a period of3 days while maintaining the above conditions in the fermentor.

At the end of the pumping of the ethyl caprylate, the pH of the broth isadjusted to between 2 and 3 using 85% phosphoric acid and the broth isthen sterilized for 15 minutes at 121° C. The broth is extracted threetimes with one-third volume of ethyl acetate. The combined extracts arewashed twice with saturated aqueous sodium chloride solution and thesolvent is evaporated.

The crude extract is then distilled at a vapor temperature of 92° C. anda pressure of 1 mm/Hg. yielding gamma octalactone having the structure:##STR105##

The yield of gamma octalactone is 7.56 grams per liter; the percentageof gamma octalactone is 58.96%; and the crude weight is 12.88 grams perliter.

EXAMPLE V Gamma Octalactone Using Mortierella Isabellina, IFO 7873##STR106##

A medium is prepared containing the following ingredients:

1.0% Peptone

0.5% Yeast Extract

0.05% TWEEN®80

10.0% Dextrose.

Into 100 ml of the above medium, a slant of Mortierella isabellina, IFO7873 is added. The inoculum of the Mortierella isabellina, IFO 7873 iscultured for a period of 3 days. At the end of the 3 day period, theculture of the Mortierella isabellina, IFO 7873 is added to 500 ml ofthe medium:

1.0% Peptone

0.5% Yeast extract

0.05% TWEEN®80

10.0% Dextrose.

500 ml Of the above culture is grown for a period of 24 hours at a pH of4.5.

Into a 9 liter fermentor operated at 27° C., 500 rpm (agitation), anaeration rate of 0.5 v/v/m and a pH of 6.5 is added the medium:

1.0% Peptone

0.5% Yeast extract

0.05% TWEEN®80

10.0% Dextrose.

500 ml of the Mortierella isabellina, IFO 7873 culture prepared above isadded to the fermentor medium with stirring and aeration as set forthabove.

Five hours after inoculation with Mortierella isabellina, IFO 7873,caprylic acid having the structure: ##STR107## is pumped into thefermentor batch at the rate of 0.27 g/hr/L. The pumping of the caprylicacid is continued for a period of 48 hours.

At the end of the 48 hour period, the pH of the broth is adjusted tobetween 2 and 3 using 85% phosphoric acid and then sterilized for 15minutes at 121° C. The broth is extracted three times with one-thirdvolume of ethyl acetate. The combined extracts are washed twice withaqueous saturated sodium chloride solution and the solvent isevaporated. The crude product is then fractionally distilled on a 12"Goodloe column at a vapor temperature of 92° C., a liquid temperature of104° C. and a pressure of 1 mm/Hg. yielding gamma octalactone having thestructure: ##STR108##

The yield of gamma octalactone is 31.44%, 4.82 grams per liter. Thecrude weight of the gamma octalactone is 15.32 grams per liter.

EXAMPLE VI Preparation of Gamma Octalactone Using MortierellaIsabellina, IFO 7873 ##STR109##

The following medium is prepared:

1.0% Peptone

0.5% Yeast extract

0.05% TWEEN®80

10.0% Dextrose.

To 100 ml of the above medium, a slat of Mortierella isabellina, IFO7873 is added and an inoculum is grown for a period of 3 days. At theend of the 3 day period, 500 ml of the medium:

1.0% Peptone

0.5% Yeast extract

0.05% TWEEN®80

10.0% Dextrose.

is inoculated with 2% of the above culture.

The 500 ml culture is grown for a period of 24 hours at a pH of 4.5.

Into a 9 liter fermentor operated at 27° C., an agitation of 500 rpm, anaeration rate of 0.5 v/v/m and a pH of 6.5, the medium:

1.0% Peptone

0.5% Yeast extract

0.05% TWEEN®80

10.0% Dextrose.

is added. With agitation and aeration, the medium in the fermentor isinoculated with the 500 ml of the above culture containing theMortierella isabellina, IFO 7873.

Twenty four hours after inoculation, pumping of the substrate, caprylicacid having the structure: ##STR110## is commenced at the rate of 0.32g/hr/L (grams per hour per liter). The pumping of the caprylic acid intothe fermentor is continued for a period of 72 hours.

At the end of the 72 hour period, the pumping ceases and the pH of thebroth is adjusted to between 2 and 3 using 85% phosphoric acid; and thenthe broth is sterilized for 15 minutes at 121° C. The broth is extracted3 times with one-third volume of ethyl acetate. The combined extractsare washed twice with saturated sodium chloride solution (aqueous) andthe solvent is evaporated.

The crude product is then fractionally distilled on a 12" Goodloe columnyielding the following fractions:

    ______________________________________                                                  Vapor        Liquid  Vacuum                                         Fraction  Temp.        Temp.   mm/Hg.                                         No.       (°C.) (°C.)                                                                          Pressure                                       ______________________________________                                        1          98/          101/   3.0                                            2         98           105     3.0                                            3         91           103     1.0                                            4         92           104     1.0                                            5         92           104     1.0                                            6         102          115     1.0                                            ______________________________________                                    

The distillation product has a percentage of gamma octalactone of55.76%; and a yield of 7.81 grams per liter. The weight of the gammaoctalactone is 14 grams per liter.

The gamma octalactone, containing a mixture of the isomers having thestructures: ##STR111## has an optical rotation of minus 28.22 degrees.

EXAMPLE VII Production of Gamma Octalactone Using MortierellaIsabellina, IFO 7873 ##STR112##

The following medium is prepared:

1.0% Enzymatic digest of soy

0.5% TASTONE®900

0.05% TWEEN®80

10.0% Dextrose.

The enzymatic digest of soy is obtained from the Deltown ChemurgicCorporation and is identified as SE-50 BT and contains:

5.1% moisture

10.59% ash

2.35% amino nitrogen

8.83% total nitrogen

55.21% protein

7.10 pH.

(SE-50 MK can be used as a replacement for SE-50 BT).

The TASTONE®900 is a brand of bakers yeast extract, spray-dried; a highclarity water soluble bakers yeast extract, spray-dried to a fine yellowtan powder having the following analysis:

65-70% protein

3-6% moisture

6.4-7.0 pH.

It is manufactured by the Universal Foods Corporation, FermentationDivision, Milwaukee, Wis.

100 ml Of the above medium is admixed with a slant of Mortierellaisabellina, IFO 7873 and an inoculum is grown for a period of threedays. The resulting inoculum is then added to 500 ml of the above mediumat a rate of 2% and the resulting inoculum is grown for a period of 24hours at a pH of 4.5.

The following medium is added to a 9 liter fermentor operated at 27° C.,agitation rate: 500 rpm and aeration rate: 0.5 v/v/m at a pH of 6.5:

1.0% enzymatic digest of soy

0.5% TASTONE®900

0.05% TWEEN®80

10.0% Dextrose.

With agitation and aeration, 500 ml of the above inoculum, grown for aperiod of 24 hours at a pH of 4.5, is added to the fermentor. Thefermentor is then operated for a period of 5 hours after which timeaddition of caprylic acid having the structure: ##STR113## is commencedat the rate of 0.32 g/hr/L. The pumping of the caprylic acid iscontinued for a period of 48 hours.

At the end of the 48 hour period, the pH of the broth is adjusted tobetween 2 and 3 using 85% phosphoric acid. The broth is then extractedthree times with one-third volume ethyl acetate and the solventevaporated. The crude product is then fractionally distilled at a vaportemperature of 92° C. and a pressure of 1 mm/Hg. yielding a compositionof matter containing gamma octalactone having the structure: ##STR114##

Examples VII(a), VII(b) and VII(c) are repeat runs of the sameexperiment. The following table shows the example, crude weight, percentgamma octalactone and the distilled product and yield:

    ______________________________________                                        Example                                                                              Crude Wt.    % g-Octalactone                                                                            Yield                                        ______________________________________                                        VII(a) 12.44 g/L    59.97         7.46 g/L                                    VII(b) 11.76 g/L    86.92        10.22 g/L                                    VII(c) 17.04 g/L    61.14         10.42 g/L.                                  ______________________________________                                    

EXAMPLE VIII Preparation of Gamma Octalactone Using MortierellaIsabellina, IFO 7884Reactions: ##STR115##

The following medium is prepared:

1.0% Peptone

0.5% Yeast Extract

0.05% TWEEN®80

10% Dextrose.

A slant of Mortierella isabellina, IFO 7884 is added to 100 ml of thismedium and the medium is cultured for a period of three days. Theresulting culture is added to 500 ml of the above medium at the rate of2%. The Mortierella isabellina, IFO 7884 is then cultured for a periodof 24 hours at a pH of 4.5.

The medium:

1.0% Peptone

0.5% Yeast Extract

0.05% TWEEN®80

10% Dextrose

is placed in a 9 liter fermentor operated at 27° C. with an agitationrate of 500 rpm and an aeration rate of 0.5 v/v/m while being maintainedat a pH of 6.5. To this medium is added 500 ml of the above inoculum ofMortierella isabellina, IFO 7884.

Five hours after inoculation, pumping of ethyl caprylate having thestructure: ##STR116## is commenced. The pumping of the ethyl caprylateis carried out at a rate of 0.31 g/hr/L and is continued for a period of72 hours.

The pH of the fermentation broth is adjusted to between 2 and 3 using85% phosphoric acid and the broth is then sterilized for 15 minutes at121° C. The fermentation broth is extracted three times with one-thirdvolume of ethyl acetate. The combined extracts are washed twice withsaturated aqueous sodium chloride solution and the solvent is thenevaporated. The crude product is then distilled on a 12" Goodloe columnyielding a product rich in gamma octalactone having the structure:##STR117## The distillation is carried out at a vapor temperature of 92°C. and a pressure of 1 mm/Hg. The yield of gamma octalactone is 11.7%;the crude weight is 8.08 grams per liter and the yield is 0.95 grams perliter.

EXAMPLE IX Preparation of Gamma Octalactone Using MortierellaIsabellina, IFO 7884 ##STR118##

The following medium is prepared:

1.0% Peptone

0.5% Yeast Extract

0.05% TWEEN®80

10% Dextrose.

A slant of Mortierella isabellina, IFO 7884 is placed into 100 ml of theabove medium and the resulting mixture is cultured for a period of 3days. At the end of the 3 day period, the resulting culture is added to500 ml of the above medium at a rate of 2%. The resulting inoculum iscultured for a period of 24 hours at a pH of 4.5.

The resulting inoculum is then added to a 9 liter fermentor which alsocontains the medium:

1.0% Peptone

0.5% Yeast Extract

0.05% TWEEN®80

10% Dextrose.

The fermentor is operated at a temperature of 27° C., an agitation rateof 500 rpm and an aeration rate of 0.5 v/v/m and is operated at a pH of6.5.

Five hours after inoculation with the Mortierella isabellina, IFO 7884,pumping of caprylic acid having the structure: ##STR119## is commencedat the rate of 0.32 g/hr/L. Pumping of the caprylic acid into thefermentor continues for a period of 48 hours.

At the end of the 48 hour period, the pH of the broth is adjusted tobetween 2 and 3 using 85% phosphoric acid, and the broth is thensterilized at 121° C. for 15 minutes. The broth is then extracted threetimes with one-third volume ethyl acetate (each time) and the solvent isevaporated. The crude product is then fractionally distilled at atemperature of 92° C. (vapor) and a pressure of 1 mm/Hg. to yield gammaoctalactone.

The yield of gamma octalactone is 33.97% and 2.63 grams per liter. Thecrude weight is 7.75 grams per liter.

EXAMPLE X

In a churn or premixer 900 kilograms of fat mixture were mixed with theaqueous phase described below. The fat mixture consisted of 25% rapeseed oil, 45% coconut oil, 20% hardened whale oil having a melting pointof 40°-42° C. and 10% hardened rape seed oil having a melting point of40°-42° C. In the fat mixture was dissolved 4 kilograms of monoglycerideand 3 kilograms of lecithin. The temperature of the fat mixture whenintroduced into the churn was about 45° C.

The aqueous phase had a temperature of about 15° C. and consisted of 115kilograms of pasteurized and cultured milk, 55 kilograms of water, 17kilograms of salt 2.8 kilograms of potato meal), 1.1 kilograms of sodiumbenzoate, 0.150 kilograms of sodium bicarbonate. 1.0 Kilograms ofdiacetyl, and 0.350 kilograms of the gamma octalactone composition ofbulked distillation Fractions 2-5 of Example VI, supra, containing gammaoctalactone.

After completion of the mixing there was added 0.750 kilograms ofvitamin oil containing 40,000 I.U. of vitamin A and 2,300 I.U. ofvitamin D₂ per gram, and 0.560 kilograms of carotene oil containing7,000 I.U. of carotene per gram. After mixing there was added an aromapreparation consisting of 2.5 kilograms of decalactone and 10 kilogramsof stearolactone dissolved in 200 kilograms of oil. The mixing wascontinued for a few minutes and the batch was then pumped to a chilledroll. The chilled emulsion was then supplied to a complector and thenput up in packets.

The result was a product having excellent taste and flavor similar tothat of butter. The flavor developed only after a few days.

EXAMPLE XI

The procedure described in Example X was repeated except that asaromatizing substances were used 0.9 kilograms of nonyllactone, 3.0kilograms of gamma octalactone, bulked distillation Fractions 2-5 ofExample VI, supra, 1.2 kilograms of undecalactone, and 12 kilograms ofstearolactone, per ton of the finished product.

EXAMPLE XII

The procedure described in Example X was repeated except that asaromatizing substances were used 1.5 kilograms of decalactone, 3.5kilograms of the gamma lactone composition of Example IX (distilled),1.0 kilograms of dodecalactone, and 10 kilograms of stearolactone, perton of finished product.

EXAMPLE XIII

The procedure described in Example X was repeated except that asaromatizing substances were used 0.25 kilograms of nonyllactone, 3.5kilograms of the composition of Example VII(b) containing gammanonyllactone, 1.6 kilograms of decalactone, 0.50 kilograms ofundecalactone, and 10 kilograms of stearolactone per ton of the finishedproduct.

EXAMPLE XIV

The procedure described in Example X was repeated except that asaromatizing substances were used 12 kilograms of stearolactone, and 8kilograms of the gamma octalactone containing composition of Example IV,supra, per ton of finished product. The taste was excellent and thefrying flavor agreeable. As in the preceding examples it was found thatpastry made with this margarine had an agreeable butter flavor andretained this flavor also after storing.

EXAMPLE XV

Artificial cream was made in the following way.

One kilogram of margarine, to which had been added emulsifying agentsfor cream whipping, was melted and had added thereto 0-0.25 milligramsof nonyllactone, 2.5 milligrams of decalactone, 3.5 grams of the gammaoctalactone-containing composition of Example III, supra, 0.5 milligramsof undecalactone and 10 milligrams of stearolactone. Two liters of milkwere then added and mixed with the margarine and the mixture was thenpassed through a homogenizer. The artificial cream thus obtained hadgood taste and was free from the extraneous taste characteristic of theordinary artificial cream.

EXAMPLE XVI Patchouli Perfume Formulation

The following mixture is prepared:

    ______________________________________                                        Ingredients         Parts by Weight                                           ______________________________________                                        Orange oil          50                                                        Bergamot oil        20                                                        Lime oil            100                                                       Neroli oil           5                                                        LYRAL ®         100                                                       (registered trademark of                                                      International Flavors &                                                       Fragrances Inc.)                                                              GALAXOLIDE ®                                                              (registered trademark of                                                      International Flavors &                                                       Fragrances Inc.)                                                              Gamma methyl ionone 20                                                        1-Acetyl-2,5,5-trimethyl-                                                                         150                                                       cycloheptane                                                                  Gamma octalactone prepared                                                                        150                                                       according to Example VI                                                       (bulked distillation                                                          Fractions 2-5)                                                                ______________________________________                                    

Bulked distillation Fractions 2-5 imparts to this patchouli formulationa sophisticated coconut topnote and tonka bean-like undertone.Accordingly, the perfume composition of Example XVI can be described as"patchouli, with coconut-like topnote and tonka bean-like undertone".

EXAMPLE XVII Preparation of Soap Compositions

One hundred grams of soap chips are produced according to Example V ofU.S. Pat. No. 4,058,487 issued on Nov. 5, 1977, the specification forwhich is incorporated herein by reference, as follows:

The sodium salt of an equal mixture of C₁₀ -C₁₄ alkane sulfonate (95%active), 40 pounds, is dissolved in a mixture of 80 pounds of anhydrousisopropanol and 125 pounds of deionized water at 150° F. In this mixtureis dissolved 10 pounds of partially hydrogenated coconut oil fatty acidsand 15 pounds of sodium mono-C₁₄ alkyl maleate, and the pH of thissolution is adjusted to 6.0 by the addition of a small amount of 50%aqueous solution of sodium hydroxide. The isopropanol is distilled offand the remaining aqueous solution is drum dried. The resulting solidactives are then blended in a chip mixture with 10 pounds of water, 0.2pounds of titanium hydroxide and 0.7 pounds of one of the perfumeingredients set forth in Table I below. The chips are then plodded intologs, cut to size and finally stamped into bars having a pH ofapproximately 6.9.

Each of the perfumed soaps produced by means of the foregoing proceduremanifests an excellent aroma as set forth in Table I, infra.

                  TABLE I                                                         ______________________________________                                        Ingredient      Fragrance Profile                                             ______________________________________                                        Mixture of compounds                                                                          A coconut aroma with Tonka                                    produced according to                                                                         bean-like undertones.                                         Example VI, bulked                                                            distillation Fractions                                                        2-5.                                                                          Perfume composition                                                                           Patchouli, with coconut-like                                  of Example XVI. topnote and Tonka bean-like                                                   undertone.                                                    ______________________________________                                    

EXAMPLE XVIII Preparation of Detergent Composition

A total of 100 grams of a detergent powder prepared according to U.S.Pat. No. 4,058,472 (the specification for which is incorporated byreference herein) and containing 5% by weight of the sodium salts of amixture of sulfonated C₁₄ -C₁₈ alkyl catechol as a surface activecomponent, the mixture being 60 parts by weight of mono-C₁₄ -C₁₈ alkylcatechol and 40 parts by weight of di-C₁₄ -C₁₈ catechol, 35% sodiumtetrapyrophosphate, 30% sodium silicate, 20% of sodium carbonate, 3% ofsodium carboxymethyl cellulose and 7% of starch is mixed with 0.15 gramsindividually with each of the aroma ingredients set forth in Table I ofExample XVII until a substantially homogeneous composition is obtained.Each of the compositions has an excellent aroma as set forth in Table Iof Example XVII.

EXAMPLE XIX Preparation of a Cosmetic Powder Composition

A cosmetic powder is prepared by mixing in a ball mill, 100 grams oftalcum powder with 0.25 grams of each of the perfume materials of TableI of Example XVII. Each of the powders has an excellent aroma as setforth in Table I of Example XVII.

EXAMPLE XX Perfumed Liquid Detergent

Concentrated liquid detergents with aromas as set forth in Table I ofExample XVII are prepared by adding 0.10%, 0.15% and 0.20% of each ofthe ingredients set forth in Table I of Example XVII. They are preparedby adding and homogeneously mixing the appropriate quantity of perfumesubstance of Table I of Example XVII in the liquid detergent. Thedetergents individually possess aromas as set forth in Table I ofExample XVII, the intensity increasing with greater concentrations ofperfume substance set forth in Table I of Example XVII.

EXAMPLE XXI Preparation of a Cologne and Handkerchief Perfume

Each of the ingredients of Table I of Example XVII is incorporatedindividually into colognes of several strengths at concentrations of2.0%, 2.5%, 3.0%, 3.5%, 4.0% and 5.0% in 75%, 80%, 85%, 90% and 95%aqueous ethanol; and into several concentrations of handkerchiefperfumes at the rate of 15%, 20% and 25% (in 80%, 85%, 90% and 95%aqueous ethanol). Distinct and definite aromas as set forth in Table Iof Example XVII are imparted to the colognes and to the handkerchiefperfumes at the several concentrations set forth above.

EXAMPLE XXII Preparation of Soap Compositions

One hundred grams of soap chips (IVORY® produced by the Proctor & GambleCompany of Cincinnati, Ohio) are admixed with one gram of each of thesubstances set forth in Table I of Example XVII, supra, untilhomogeneous compositions are obtained. In each of the cases, thehomogeneous compositions are heated under 3 atmospheres pressure at 180°C. for a period of three hours and the resulting liquids are placed intosoap molds. The resulting soap cakes, on cooling, manifest excellentaromas as set forth in Table I of Example XVII.

EXAMPLE XXIII Preparation of Solid Detergent Compositions

Detergents are prepared from the following ingredients according toExample I of Canadian Patent No. 1,007,948, the specification for whichis incorporated by reference herein:

    ______________________________________                                        Ingredients         Parts by Weight                                           ______________________________________                                        NEODOL ® 45-11  12                                                        (a C.sub.14 -C.sub.15 alcohol                                                 ethoxylated with 11 moles                                                     of ethylene oxide)                                                            Sodium carbonate    55                                                        Sodium citrate      20                                                        Sodium sulfate, water brighteners                                                                 q.s.                                                      ______________________________________                                    

This detergent is a "phosphate-free" detergent. A total of 100 grams ofsaid detergent is admixed with 0.10, 0.15, 0.20 and 0.25 grams of eachof the substances set forth in Table I of Example XVII, supra. Each ofthe detergent samples has an excellent aroma as indicated in Table I ofExample XVII.

EXAMPLE XXIV Preparation of Drier-Added Fabric Softener Article

Utilizing the procedure of Example I at column 15 of U.S. Pat. No3,632,396 the specification for which is incorporated by referenceherein, a non-woven cloth substrate useful as a drier-added fabricsoftening article of manufacture is prepared wherein the substrate,substrate coating and outer coating and the perfume material are asfollows:

1. a water "dissolvable" paper ("Dissolvo Paper") as the substrate;

2. ADOGEN®448 (melting point about 140° F.) as the first substratecoating; and

3. an outer coating having the following formulation (melting pointabout 150° F.);

57% C-₂₀ -C₂₂ HAPS;

22% isopropyl alcohol;

20% antistatic agent; and

1% of one of the perfumery substances set forth in Table I of ExampleXVII, supra.

Fabric softening compositions containing the substances as set forth inTable I of Example XVII, supra, essentially consist of a substratehaving a weight of about 3 grams per 100 square inches; a substratecoating weighing about 1.85 grams per 100 square inches of substrate;and an outer coating weighing about 1.5 grams per 100 square inches ofsubstrate are prepared thereby providing a total aromatized substrateand outer coating weight ratio of about 1:1 by weight of the substrate.

The aromas as set forth in Table I of Example XVII, supra, are impartedin a pleasant manner to the head space in a drier on operation thereofusing the said drier-added fabric softening non-woven fabric by addingto the drying cycle.

As stated above in the case of fabric softener articles, the entire U.S.Pat. No. 3,632,396 is incorporated by reference herein. Thus, all of thearticles of U.S. Pat. No. 3,632,396 acting as fabric softening articlesin said U.S. Patent may be perfumed in their outer coating with from0.25% up to 5% by weight of each of the perfuming substances of Table Iof Example XVII, supra.

EXAMPLE XXV Hair Preparation

A "soft-feel, good-hold" hair spray is produced containing the followingingredients:

    ______________________________________                                        Ingredients          Parts by Weight                                          ______________________________________                                        Polyvinylpyrollidone/vinyl                                                                         4.00                                                     acetate "E-735 Copolymer"                                                     manufactured by the GAF                                                       Corporation of New York, N.Y.                                                 Anhydrous ethanol    70.90                                                    Dioctyl sebecate     0.05                                                     Benzyl alcohol       0.05                                                     "Propellant A-46" manufactured                                                                     24.95                                                    by the GAF Corporation of                                                     New York, N.Y.                                                                Fragrance ingredient as set                                                                        0.05                                                     forth in Table I of Example XVII,                                             supra.                                                                        ______________________________________                                    

The PVP/VA copolymers re first dissolved in alcohol and all otheringredients are added until uniform. The propellant is then pressurizedand used as an aerosol. The resulting hair sprays each have pleasantaromas as set forth in Table I of Example XVII, supra.

EXAMPLE XXVI Scouring Cleanser Composition

A scouring cleanser composition is prepared in accordance with Example Iat columns 11 and 12 of U.S. Pat. No. 4,193,888 issued on Mar. 18, 1980,the specification for which is incorporated by reference herein. To thiscomposition, the substances set forth in Table I of Example XVII, supra,are added at the level of 0.25% as set forth in the table in saidExample I of U.S. Pat. No. 4,193,888 yielding an aroma on using saidcleanser in ordinary circumstances which is quite pleasant and describedin Table I of Example XVII, supra.

EXAMPLE XXVII

A fabric softening article prepared substantially as set forth inExample VIII of Canadian Patent No. 1,069,260, the specification forwhich is incorporated by reference herein, is prepared containing 0.21%by weight of a perfuming substance as set forth in Table I of ExampleXVII, supra, and yielding on use in a drier, a faint aroma as set forthin Table I of Example XVII, supra.

EXAMPLE XXVIII Tobacco Flavor Formulations

Cigarettes are produced using the following tobacco formulations:

    ______________________________________                                        Ingredients     Parts by Weight                                               ______________________________________                                        Bright          40.1                                                          Burley          24.9                                                          Maryland        1.1                                                           Turkish         11.6                                                          Stem (flue cured)                                                                             14.2                                                          Glycerine       2.8                                                           H.sub.2 O       5.3                                                           ______________________________________                                    

At the rate of 0.2%, the following tobacco formulation is applied to allof the cigarettes produced with the above tobacco formulation:

    ______________________________________                                        Ingredients     Parts by Weight                                               ______________________________________                                        Ethyl butyrate  0.05                                                          Ethyl valerate  0.05                                                          Maltol          2.00                                                          Cocoa extract   26.00                                                         Coffee extract  10.00                                                         Ethyl alcohol (95%)                                                                           20.00                                                         H.sub.2 O       41.900                                                        ______________________________________                                    

To portions of 50% of the cigarettes at levels of 10 and 20 ppm, theoctalactone-containing composition of Example V (distilled) is added.These cigarettes are hereinafter called "experimental" cigarettes. Thecigarettes without the octalactone composition are hereinafter called"control" cigarettes. The control and experimental cigarettes are thenevaluated by paired comparison and the results are as follows:

(a) In aroma, the experimental cigarettes are all found to be morearomatic with Turkish tobacco-like nuances;

(b) In smoke flavor, the experimental cigarettes are all found to bemore aromatic, more sweet with Turkish tobacco, oriental-like nuancesthan the control cigarettes.

The experimental cigarettes containing the mixture of lactones are foundto be fruity and have pleasant aesthetically pleasing fruity notes inaddition.

EXAMPLE XXIX Pudding

At the rate of 0.8 ppm, the composition containing gamma octalactone ofExample VI, bulked distillation Fractions 2-5 is added to a ROYAL®butterscotch pudding composition. Pleasant aesthetically pleasingcoconut nuances were added to the butterscotch pudding as a result ofthe use of the octalactone composition. Without the octalactonecomposition no such coconut nuances are added. A panel of 10 individualsprefers the octalactone-containing butterscotch pudding.

What is claimed is:
 1. A process for preparing a composition containinga significant amount of optically active gamma octalactones having thestructures: ##STR120## and in addition a mixture of compounds having thestructures: ##STR121## comprising the sequential steps of: (i) carryingout a fermentation of a compound having the structure: ##STR122## in thepresence of an organism selected from the group consisting of:Mortierella isabellina, ATCC 44583;Mortierella isabellina, ATCC 38063;Syncephalastrum racemosum, NRRL A-5889; Mortierella isabellina, IFO7884; Mortierella ramanniana var. anguliospora, IFO 8187; Mortierellaisabellina, CBS 221.29; and Mortierella isabellina, IFO 7873;Mortierella ramanniana var. ramanniana, CBS 112.08 Mortierellaramanniana var. ramanniana, CBS 478.63 Choanephora cucurbitarum, NRRL2744 Mortierella isabellina, IFO 8183 Mortierella isabellina, IFO 8309Mortierella nana, IFO 8794 Mortierella isabellina, IFO 7824 Mortierellavinacea, IFO 6738 in aqueous media, at a temperature between about 18°C. up to about 31° C., at a pH in the range of from about 3 up to about9, for a period of time of from 40-200 hours under aerobic conditionsmaintaining the dissolved oxygen content from 20% to 100% by weightwhereby a compound defined according to the structure: ##STR123## isproduced according to the reaction: ##STR124## wherein R is ethyl orhydrogen; (ii) effecting lactonization of the resulting gamma hydroxyoctanoic acid by heating the said acid according to the reaction:##STR125## (iii) distilling the resulting mixture at a vapor temperatureof 91°-98° C. and a pressure of 1-3 mm/Hg. to yield a mixture ofcompounds having the structures: ##STR126##
 2. The process of claim 1wherein R is hydrogen.
 3. The process of claim 1 wherein R is ethyl. 4.The process of claim 1 wherein the resulting hydroxy octanoic acid has astructure selected from the group consisting of: ##STR127##
 5. Theproduct produced according to the process of claim
 1. 6. The process ofclaim 1 wherein process step (i) is carried out at 25°-27° C. for aperiod of time of from 48-72 hours at a pH of from 4.5-6.5 and processstep (ii) is carried out at a pH of from 2-3 at 121° C. for a period oftime of 15 minutes.